7 dehydrocholesterol

Drosophila pachea flies were retrieved from the San Diego Drosophila stock center (15090–1698.02) and maintained for about 40 generations at 25 °C in vials with 10 ml of standard Drosophila medium, mixed with 200 μg of 7-dehydrocholesterol (Sigma) dissolved in 40 μl of ethanol [67 (link), 68 (link)]. In this stock, about 20 % of the males possess short and bilaterally symmetric epandrial lobes (symmetric males) [43 ]. All flies came from the 15090.1698.02 stock except a few symmetric mutant males which came from a selection line that was derived from the 15090.1698.02 stock by selecting symmetric males for several generations in order to increase the proportion of symmetric males.

Water (HPLC grade), methanol
(MeOH, HPLC grade), acetonitrole (ACN, HPLC grade), isopropanol (IPA,
HPLC grade), stigmasterol, ergosterol, and 7-dehydrocholesterol were
purchased from Sigma-Aldrich (MO, USA). Hydroxylamine-O-sulfonic acid (HOSA) was purchased from Combi-Blocks (CA, USA).
Hexafluoro-2-propanol (HFIP), cholesterol, and β-sitosterol
were purchased from Fisher Scientific (NH, USA). Pyridine was purchased
from Millipore Sigma (MA, USA). Bis[rhodium(α,α,α′,α′-tetramethyl-1,3-benzenedipropionic
acid)] (Rh₂(esp)₂) was from AmBeed (IL, USA).
All solvents and chemicals were used without further purification.

Either LB- (25 g/L, Becton–Dickinson) or TB- (24 g/L yeast extract, 12 g/L tryptone, 0.4% glycerol, 100 mM potassium phosphate buffer, pH 7.4) media were used for the cultivation of B. megaterium. For the preculture, 50 mL of medium either containing 10 µg/mL tetracycline (cells harboring a variant of pSMF2.1) or 10 µg/mL chloramphenicol (cells harboring a variant of pSMF3) were inoculated with cells from an agar plate or glycerol stock. For the main culture, 50 mL of medium containing 10 µg/mL of the required antibiotic were inoculated with 500 µL of the preculture. The main culture was grown until an optical density of ~0.4 was reached, protein expression was induced by addition of 0.25 g xylose dissolved in 1 mL distilled water. For whole-cell conversion experiments, substrates were dissolved in a 45% 2-hydroxypropyl-β-cyclodextrin/4% Quillaja saponin-solution. 2.5 mL of the solution were added to the culture directly after protein induction. A final substrate concentration of 300 µM was used for all conversion experiments. Cholesterol from sheep wool (purity ≥99%), 7-dehydrocholesterol (≥98%), β-sitosterol from plant extracts (purity ≥70%, containing residual campesterol), 2-hydroxypropyl-β-cyclodextrin (average molecular weight ~1,460) and Quillaja saponin (Sapogenin content ≥10%) were purchased from Sigma-Aldrich.

For the rescue experiments, 20 mg of dry yeast was mixed with 38 μl H₂O and 2 μl ethanol or supplemented with 2 μl of the following sterols dissolved in ethanol: cholesterol (Wako; 150 mg/ml), 7-dehydrocholesterol (Sigma; 150 mg/ml), 5β-ketodiol (kindly gifted from Yoshinori Fujimoto, Tokyo Institute of Technology; 150 mg/ml) and 20-hydroxyecdysone (Sigma; 50 mg/ml). We crossed ouib²⁹/TM3 Ser¹GMR2 Act-GFP flies with ouib⁷⁴/TM3 Ser¹GMR2 Act-GFP flies. Eggs were laid on grape plates with yeast pastes at 25°C for 12 hours. At 36 hours AEL, 50 hatched GFP negative (ouib²⁹/ouib⁷⁴) first instar larvae were transferred to the yeast paste on grape plates and kept at 25°C. Every 24 hours, developmental stages were scored by tracheal morphology as previously described [22 (link)].

β-sitosterol (>98% purity), campesterol (>65% purity), desmosterol (>85% purity), ergosterol (>95% purity), cholesterol (>99% purity), 7-dehydrocholesterol (>99% purity), ecdysone (>90% purity), and 20-hydroxyecdysone (>93% purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). cholesterol-3,4-₁₃C₂ (>99% purity) was purchased from C/D/N Isotopes, Inc (Pointe-Claire, Quebec, Canada). Stigmasterol (>99% purity) was purchased from Tama Biochemical Co (Tokyo, Japan).

Cholesterol, stigmasterol, β-sitosterol, 7-dehydrocholesterol, lathosterol (5α-cholest-7-en-3β-ol), squalene, campesterol, brassicasterol, desmosterol, lanosterol, fucosterol, cycloartenol, and 5α-cholestane (internal standard) were acquired from Sigma-Aldrich (Saint-Quentin-Fallavier, France), cycloartanol and cycloeucalenol from Chemfaces (Wuhan, China), and zymosterol from Avanti Polar Lipids (Alabaster, United States). The C7-C40 Saturated Alkanes Standards were acquired from Supelco (Bellefonte, United States). Reagents used for extraction, saponification, and derivation steps were n-hexane, ethyl acetate, acetonitrile, methanol (Carlo ERBA Reagents, Val de Reuil, France), (trimethylsilyl)diazomethane, toluene (Sigma-Aldrich) and N,O-bis(trimethylsilyl)trifluoroacetamide with trimethylcholorosilane [BSTFA:TMCS (99:1); Supelco].